Journal of Nematology 38(3):362375. 2006. The Society of Nematologists 2006.

The Comparative Cellular Architecture of the Female Gonoduct Among Tylenchoidea (Nematoda: Tylenchina)
W. Bert, M. Claeys, G. Borgonie
Abstract: The cellular architecture of the female gonoduct of 68 nematode populations representing 42 species belonging to Tylenchidae, Belonolaimidae, Hoplolaimidae and Meloinema is shown to have an overall similarity in cellular gonoduct structure. The oviduct consists of two rows of four cells; the spermatheca is comprised of 10 to 20 cells, and the uterus cells, except in the case of Psilenchus, are arranged in four (Tylenchidae) or three (Belonolaimidae, Hoplolaimidae and Meloinema) regular rows. Although the genus Meloinema is classied within Meloidogynidae, its spermatheca is clearly hoplolaimid-like and lacks the spherical shape with lobe-like protruding cells typical of Meloidogyne. Detailed morphology of expelled gonoducts may provide a valuable character set in phylogenetic analysis, and the cellular morphology of the spermatheca appears to be a distinguishing feature at species level, especially in the genera Tylenchus and Geocenamus. Ultrastructural data on the oviduct-spermatheca region of Meloidogyne incognita complement light-microscopic (LM) results. The combination of LM of expelled organs and transmission electron microscopy (TEM) on selected sections is put forward as a powerful tool to combine three-dimensional knowledge with ultrastructural detail. Key words: Belonolaimidae, electron microscopy, gonoduct, Hoplolaimidae, Meloinema, morphology, taxonomy, TEM, Tylenchidae, ultrastructure

The female reproductive system has been shown to be important in nematode systematics (Geraert, 1981, 1983), and the cellular morphology with respect to number and spatial arrangement of cells seems to be specic and constant for many nematodes species. Detailed examinations of the female reproductive system of free-living and plant-parasitic, as well as insectparasitic, nematodes have been undertaken (Geraert, 1973, 1976; Geraert et al., 1980a, 1980b; Chizhov, 1981; Chizhov and Swiliam, 1986; Chizhov and Berezina, 1988a, 1988b). Recently, Bert et al. (2002, 2003) focused on the gonoduct structure of endoparasitic nematode families Pratylenchidae, Heteroderinae and the genus Meloidogyne. Analyzing the structure of expelled gonoducts explores informative new morphological comparative characters. Although molecularbased Tylenchoidea phylogenies are advancing (Subbotin et al., 2006), morphological characters may provide a valuable independent character set to be mapped on molecular-based branches or to construct total-evidence phylogenies. Whereas the vastly superior resolution of an electron microscope has been shown to provide an improved phylogenetic signal (e.g., Zhang and Baldwin, 2000; Baldwin et al., 2001), the herein presented dissection technique generates new morphological data on a relatively less time expensive base compared to transmission electron microscopy. Furthermore, additional morphological characters are essential for a more substantiated differentiation of

plant-parasitic nematode taxa which often posses a deceptively similar anatomical pattern. The objectives of this study were to achieve (i) a lightmicroscopic detailed gonoduct analysis of 68 populations of 42 species of Tylenchidae, Belonolaimidae and Hoplolaimidae in order to complete the knowledge of the female gonoduct structure within the Tylenchoidea, and to develop (ii) a complementary ultrastructural study of the oviduct-spermatheca region of Meloidogyne incognita for improved insight in our LMobtained data and to clarify relationships. Material and Methods Nematode species were obtained from soil samples or cultures (Table 1). Extraction and examination of the female reproductive system was based on the method of Geraert (1973), i.e., bisecting the specimen at the vulva region with a small scalpel-induced expulsion of gut and gonad. Nematodes were further processed without removing nonreproductive tissue to avoid damage. The gonoduct expulsion procedure was repeated until at least 20 preparations could be observed for each population (except as noted). Preparations were either stained with acetic orcein (UCB, Leuven, Belgium, 2% aqueous solution of orcein in acetic acid) or observed directly in temporary mounts with the light microscope. By staining, a stronger differentiation of the nuclei is achieved, whereas without staining, the general cell morphology is better preserved. The cellular morphology of the ovary was only partly (or not) studied with light microscopy, as it turned out to be difficult to observe all structures; only the ripening zone of the ovary contains distinct cells which could be visualised with the techniques used here. Two types of light microscopes were used during this study: a Reichert Zetopan (Reichert-Leica, Vienna, Austria) and an Olympus BX 51 DIC (Olympus optical, Tokyo, Japan). Measurements and illustrations were prepared using a camera lucida; the drawings were prepared using Illus-

Received for publication March 31, 2006 Nematology Unit, Department of Biology, Ghent University, Belgium. This work was supported by a research equipment grant of the Fund for Scientic ResearchFlanders, Belgium (Grant 1.5.090.05 and 0194.03). The authors thank D. G. Kim, E. Pourjam, P. De Ley, I. Tandingan De Ley, G. Elbadri and G. Karssen for their generous supply of specimens or help in nding sample localities and W. Decraemer for critical reading and helpful comments. The anonymous reviewers are acknowledged for their valuable suggestions. Wim.Bert@UGent.be This paper was edited by Zafar Handoo

362

Tylenchoidea gonoduct: Bert et al. 363

TABLE 1. List of species studied (and their authorities), description of sampling site or culture and location
Species Sampling site/Population origin Location/Source

Sand dune covered with Hippophae rhamnoides L. (sea buckthorn) Sand mound on fallow, vegetation dominated by Urtica dioica L. and Glechoma hederaceae L., loamy sand T. arcuatus Siddiqi, 1963 Apple orchard, sandy loam soil Lawn in the vicinity of a willow tree (Salix matsudana Koidz), sandy loam soil Mosses on rocks Filenchus vulgaris Grassland on former dump site (Brzeski, 1963a) Lownsbery & Lawn in the vicinity of a willow tree Lownsbery, 1985 (Salix matsudana Koidz), sandy loam soil F. thornei (Andra ssy, 1953) Andra ssy, 1963 Apple orchard, sandy loam soil F. quartus (Szczygiel, 1969) Lownsbery & Lawn in the vicinity of a willow tree Lownsbery, 1985 (Salix matsudana Koidz), sandy loam soil F. facultativus Meadow in the vicinity of apple tree (Szczgiel, 1969) Raski & Geraert, 1987 Filenchus cf. facultativus (Szczygiel, 1969) Rhizosphere of willow above stony drain Raski & Geraert, 1987 Filenchus cf. orbus Canal bank (Andra ssy, 1954) Meyl, 1961 Filenchus cf. terrestris Grassland Raski & Geraert, 1987 Coslenchus costatus Moervaart canal bank (de Man, 1921) Siddiqi, 1978 Lawn in the vicinity of a willow tree (Salix matsudana Koidz), sandy loam soil C. polonicus Brzeski, 1982 Wetland, sandy loam soil with a high peat content C. andrassyi Brzeski, 1987 Border of a football pitch Lawn, sandy loam soil Coslenchus cf. polygyrus Bajaj & Bhatta, Potato eld 1983 Aglenchus agricola (de Man, 1880) Meyl, Lawn in the vicinity of a willow tree (Salix 1961 matsudana Koidz), sandy loam soil Apple orchard, sandy loam soil Football pitch Lawn, loamy sand soil Basiria duplexa (Hagemeyer & Allen, Lawn, light sandy loam soil 1952) Geraert, 1968 B. graminophila Siddiqi, 1959 Potato eld Grassland on former dump site Rhizosphere of Brassica rapa L. (rape) B. gracilis (Thorne, 1949) Siddiqi, 1963 Grassland on former dump site Boleodorus thylactus Thorne, 1941 Lawn in the vicinity of a willow tree (Salix matsudana Koidz), sandy loam soil unknown Neopsilenchus magnidens (Thorne, 1949) Apple orchard, sandy loam soil Thorne & Malek, 1968 Psilenchus aestuarius Andra ssy, 1962 Grassland on former dump site Cephalenchus leptus Siddiqi, 1963 Apple orchard, sandy loam soil Tropical rainforest C. hexalineatus (Garaert, 1962) Geraert & Uncovered soil in the vicinity of Hedera helix L. Goodey, 1964 (common ivy) and Betula sp. (birch) Geocenamus brevidens (Allen, 1955) Grassy riverbank, vegetation dominated by Brzeski, 1991 Arrhenatherion elatius (L.) Presl and Holcus lanatus L. Geocenamus nothus (Allen, 1955) Brzeski, Grassy riverbank, vegetation dominated by 1991 Arrhenatherion elatius and Holcus lanatus Pistacio Geocenamus quadrifer (Andra ssy, 1954) Grassy riverbank, vegetation dominated by Brzeski, 1991 Arrhenatherion elatius and Holcus lanatus Geocenamus cf. nurserus (Eroshenko & Rhizosphere of willow above stony drain Volkova, 1987) Fortuner & Luc, 1989 Tylenchorhynchus dubius (Bu tschli, 1873) Corn eld Tylenchus davainei Bastian, 1865 T. elegans de Man, 1876

Knokke, Belgian coast Ghent, Belgium

Vliermaal, Belgium Botanical garden Ghent University, Belgium Idem Ghent, Belgium Botanical garden, Ghent University, Belgium Vliermaal, Belgium Botanical garden, Ghent University, Belgium Pleaux, France 3413.508N, 11703.361W, Route 18, California, US Ghent, Belgium Bourgoyen-Ossemeersen, Ghent, Belgium Lokeren, Belgium Botanical garden Ghent University, Belgium Bourgoyen-Ossemeersen, Ghent, Belgium Heusden, Destelbergen, Belgium Sint-Amands, Antwerp province, Belgium Schorisse, Flemish Ardennes, Belgium Botanical garden Ghent University, Belgium Vliermaal, Belgium Heusden, Destelbergen, Belgium Geel, Antwerp province, Belgium Sint-Amands, Antwerp province, Belgium Schorisse, Flemish Ardennes, Belgium Ghent city, Belgium Latitpur district, Nepal Ghent city, Belgium Botanical garden, Ghent University, Belgium Spain Vliermaal, Limburg province, Belgium Ghent city, Belgium Vliermaal, Limburg province, Belgium Kakamega, Kenya Landegem, East-Flanders province, Belgium Moervaart canal, Lokeren, Belgium

Moervaart canal, Lokeren, Belgium Kerman, Irana Moervaart canal, Lokeren, Belgium 3413.508N, 11703.361W, Route 18, California, US Eksaarde, Lokeren, Belgium

364 Journal of Nematology, Volume 38, No. 3, September 2006

TABLE 1. Continued
Species Sampling site/Population origin Location/Source

Riverbank, vegetation dominated by Crepis capillaries (L.) Wallr., Plantago major L., Trifolium repens L. and Poa annua L. Light sandy loam soil T. microphasmis Loof, 1960 Ammophila arenaria (L.) Link in sand pot Grassy riverbank T. ventralis (Loof, 1963) Fortuner & Luc, Ammophila arenaria (L.) Link in recipient lled 1987 with sand T. maximus Allen, 1955 Grassy riverbank Nagelus obscurus (Allen, 1955) Powers, Phragmites australis (reed) Baldwin & Bell, 1983 Amplimerlinius icarus (Wallace & Greet, Lawn in the vicinity of a willow tree (Salix 1964) Siddiqi, 1976 matsudana Koidz) Apple orchard, sandy loam soil Helicotylenchus cf. dihystera (Cobb, 1893) Unknown Sher, 1961 H. pseudorobustus (Steiner, 1914) Golden, Wetland 1956 H. varicaudatus Yuen, 1964 Grassy riverbank, vegetation dominated by Arrhenatherion elatius and Holcus lanatus H. canadensis Waseem, 1961 Riverbank, vegetation dominated by Crepis capillaries (L.) Wallr., L., Plantago major L., Trifolium repens L. and Poa annua L., light sandy loam soil Rotylenchus goodeyi Loof & Oostenbrink, Riverbank, vegetation dominated by Crepis 1958 capillaries (L.) Wallr., L., Plantago major L., Trifolium repens L. and Poa annua L., light sandy loam soil Ammophila arenaria (L.) Link in sand pot Unknown Rotylenchus uniformis Ligustrum hedge (Ligustrum vulgare L.) Scutellonema bradys (Steiner & Le Hew, Tropical plants in greenhouse 1933) Andra ssy, 1958 Hoplolaimus aegypti Shafee & Koura, 1970 Banana Rotylenchulus reniformis Linford & Original host: carnation, Inida Oliveira, 1940 Cultured on beet Meloinema odesanens Kim, Vovlas, Choi & Mountain tree (Tilia amurensis Rupr.) Lee, 2005 Meloidogyne incognita (Kofoid and White Tomato culture 1919) Chitwood, 1949

Filipjev 1936

Canal Roeselare-Leie, Ingelmunster, Belgium Heteren, The Netherlandsb Moervaart canal, Lokeren, Belgium Heteren, The Netherlandsb Moervaart canal, Lokeren, Belgium Bourgoyen-Ossemeersen, Ghent, Belgium Botanical garden Ghent University, Belgium Vliermaal, Belgium Khartoum, Sudand Bourgoyen-Ossemeersen, Ghent, Belgium Moervaart canal, Lokeren, Belgium Canal Roeselare-Leie, Ingelmunster, Belgium

Canal Roeselare-Leie, Ingelmunster, Belgium Heteren, The Netherlandsb Sennar state, Sudand Gentbrugge, Belgium CABI Bioscience, Egham, UKc Hantop, Sudad Wageningen, The Netherlandsc Gwangweon Province, Koreaf Wageningen, The Netherlandse

Samples obtained from: a E. Pourjam, Tarbiat Modares University, Tehran, Iran b Netherlands Institute of Ecology, Heteren, The Netherlands c P. De Ley, Department of Nematology, University of California, Riverside, USA d G. Elbadri, Crop Protection, Wad Medani, Sudan e G. Karssen, Plant Protection Service, Wageningen, The Netherlands f D. G. Kim, Department of Agricultural Environment, Gyeongbuk Agricultural Technology Administration, Daegu, Korea

trator 10.0 software (Adobe Systems, Mountain View, CA, USA). The morphology was also recorded as video clips that mimic multifocal observation through a light microscope following the Video Capture and Editing procedures developed by De Ley and Bert (2002). The resulting virtual specimens are available on the web at: http://www.nematology.ugent.be/VCE.htm. The terminology of the reproductive system used here is based on Geraert (1983), who followed Chitwood and Chitwoods (1950) interpretation. A genital branch, as occurs in a in a didelphic or monodelphic genital system, consists of an ovary (= gonad) and gonoduct. The ovary consists of three functional zones, namely, the germinal zone, the growth zone and the ripening zone. The oviduct is the constricted region between ovary and spermatheca. The term uterus is here restricted to the eggshell-formation region (also demarcated as columnar region if distinct uterus cell

rows are visible) of the gonoduct; a constriction may be present between the uterus and the uterine sac. The uterine sac follows the uterus and terminates in the vagina, which is connected to the vulva. The classication of the studied organisms is largely based on De Ley and Blaxter (2002), but below family level on a reappraisal of Tylenchina (see Maggenti et al., 1987). Expelled gonoducts of Meloidogyne incognita were examined by transmission electron microscopy (TEM). Excised reproductive systems from 30 specimens were transferred to ice-cooled Karnovskys (1965) xative. The xation, embedding, sectioning and TEM procedures are as previously described for in toto specimens (Bert et al., 2003). Results A summary of the results is presented in Table 2. Tylenchidae (Figs. 13): The oviduct of members of

Tylenchoidea gonoduct: Bert et al. 365

TABLE 2. The cellular composition of oviduct (number of cell rows, number of cells per row), spermatheca (cell number, structure) and uterus (number of cell rows, number of cells per row) is listed for each species studied. When data are unknown or uncertain, a question mark (?) is printed.
Oviduct Cell rows cells per row Spermatheca Cell number offset pouch Uterus Cell rows cells per row

Cell number

rley, 1880 Tylenchidae O 2 34 1214 (+2) 23 14 (+2) 2 4 (3) 12 (+2) 23 14 23 1416 2 3 (4) 14 23 14 23 14 (16) 24 1516 23 14 24 14 (16) 24 14 24 14 24 1012 2 4 (3) (12)14(16) 2 5(6) 16 25 16 25 16 25 16 25 16 25 1820 2 57 12 2 57 12 Belonolaimidae Whitehead, 1960 24 14 Geocenamus nothusa G. quadrifera 24 12 24 1214 G. cf. nurserusa G. brevidensa 24 12 24 ? G. microdorusa Tylenchorhynchus dubiusa 24 12 (14) 24 1214 T. microphasmisa T. ventralisa 24 1214 24 810 T. maximusa a Nagelus obscurus 2 4 (5) 1214 24 1214 Amplimerlinius icarusa Hoplolaiminae Filipjev, 1934 Helicotylenchus varicaudatusa 24 12 24 12 H. canadensisa 24 12 (+2) H. pseudorobustusa H. cf. dihysteraa 24 12 (+2) 24 12 (14) Rotylenchus uniformisa R. goodeyi a 24 12 (14) Scutellonema bradysa 24 1214 Hoplolaimus aegyptia 24 912? Rotylenchulinae Husain & Khan, 1967 24 1217? Rotylenchulus reniformisa Meloinema Choi & Geraert, 1974 M. odesanensa 24 1214 Meloidogyne Go ldi, 1892 (see also Bert et al., 2003) Tylenchus davainei T. elegans T. arcuatus Filenchus vulgaris F. thornei F. quartus F. facultativus Filenchus cf. facultativus Filenchus cf. terrestris Filenchus cf. orbus Coslenchus polonicus C. andrassyi C. cf. polygyrus C. costatus Aglenchus agricola Basiria graminophila B. duplexa B. gracilis Boleodorus thylactus Neopsilenchus magnidens Psilenchus aestuariusa Cephalenchus leptus C. hexalineatus M. incognita 24 16 lobe-like cells with interlaced cell boundaries

44 44 44 44 4 (irregular) 4? 44 44 4? 44 4? ? 4 78 4 (irregular) 56 4 (irregular) ? 4 5 (6) 44 4 (irregular) 4 (5) 4 (irregular) 45 4 (irregular) 4 (5) 4 4 (irregular) 810 4? 3855? 4 78 4 78 Spermatheca structure 4 lobes 3 45 offset 3 37 spherical 3 57? spherical 3 45 2 lobes 34 round-oval 3 45 oval, 2 compartments 35 oval 3 56 oval 3 79 offset 34 bell-shaped 3? maximally offset maximally offset offset offset oval oval ovalbell-shaped unclear elongated? oval 34 34 34 3 4? 3 4 (5) 3 4 (3) 3? 3? 3? ? 3 long rows

57 4 610 ? 68 710 68 68 710 710 710 412 >7

spherical

3 long rows

didelphic reproductive system

the family Tylenchidae comprises two rows of three to seven cells. The oviduct of Tylenchus, Filenchus, Coslenchus and Aglenchus is composed of two rows of three or four cells. In Basiria, Boleodorus, Neopsilenchus and Psilenchus, ve (exceptionally six) cells per row are present

with the most proximal oviduct cells usually being slightly larger; Cephalenchus is characterized by a longer and slightly bent oviduct that comprises two rows of ve, six or seven cells. The uterus cells, except for Psilenchus aestuarius, are

366 Journal of Nematology, Volume 38, No. 3, September 2006 arranged in four regular rows (= quadricolumella) each with four to ten cells; the nuclei of these cells are in most cases distinctly larger than the spermatheca. Six tightly packed cells between the quadricolumella and uterine sac, forming a sphincter-like structure, were observed for Tylenchus arcuatus. The genital branches of the didelphic reproductive system of Psilenchus aestuarius are considerably longer. The uterus cells do not form a quadricolumella, but are arranged in irregular rows, each comprising 38 to 55 cells. Also, Coslenchus andrassyi, C. cf. polygyrus and Neopsilenchus display a partially irregular arrangement of the uterus cells. The spermatheca shows several differences in cellular architecture within the Tylenchidae. The spermatheca of Tylenchus (Fig. 1A-C) comprises 12 to 14 cells and is axial in T. arcuatus, while partially offset in T. davainei

Fig. 1. The cellular architecture of oviduct, spermatheca and uterus of Tylenchus spp. and Filenchus spp. A) T. arcuatus. B) T. davainei. C) T. elegans. D) F. vulgaris from dump site. E) F. vulgaris from botanical garden. F) F. thornei. G) F. orbus. H) F. facultativus. I) F. cf. terrestris. J) F. cf. facultativus. ova.: proximal end of ovary; ovi.: oviduct; sp.: spermatheca; ut.: uterus; con.: constriction between uterus and uterine sac. Scale bars = 10 m

Tylenchoidea gonoduct: Bert et al. 367 and T. elegans with the offset pouch composed of ve to seven cells and four cells, respectively. The spermatheca is connected to the uterus by two large cells. The spermatheca of Filenchus species (Fig. 1D-J) is axial (F. quartus), slightly offset (F. thornei), with a distinctly offset pouch comprising six to eight cells (F. facultativus, F. cf. terrestris, F. cf. orbus) or with an oblong offset pouch encompassing seven to 10 cells (F. cf. facultativus ). Two large, rounded cells connect the spermatheca with the uterus in F. quartus, F. cf. terrestris and F. cf. orbus. Coslenchus polonicus, C. andrassyi and C. cf. polygyrus display a similar spermatheca (Fig. 2A-D), comprised of 14 cells (exceptionally 16 cells are observed in C. polonicus). The spermathecae of these Coslenchus species consist of a distinctly offset pouch, composed of seven to 10 cells, and an axial part, encompassing two large cells that are nearly as long as the offset sac. The majority of the examined C. andrassyi

Fig. 2. The cellular architecture of oviduct, spermatheca and distal part uterus of Coslenchus spp., Agelenchus and Cephalenchus spp. A) Coslenchus costatus. B) C. cf. polygyrus. C) C. andrassyi. D) C. polonicus. E) Aglenchus agricola, including LM photographs. F) Cephalenchus leptus from apple orchard. G) C. hexalineatus. ovi.: oviduct; sp.: spermatheca; ut.: uterus. Scale bars = 10 m.

368 Journal of Nematology, Volume 38, No. 3, September 2006 specimens display two particularly small cells that connect spermatheca to uterus. Coslenchus costatus is characterized by an axial spermatheca that is indistinctly demarcated from adjacent regions of the gonoduct and comprises only 10 to 12 cells. The spermatheca of Aglenchus agricola (Fig. 2E) generally comprises 14 cells; though cell numbers between 12 and 16 were also observed, their cell boundaries are often only vaguely visible. The size of the offset pouch (consisting of four to 12 cells) shows a remarkable intraspecic variability in this species. The genus Cephalenchus (Fig. 2F,G) is characterized by an axial spermatheca comprised of 12 cells; the spermatheca cells of C. leptus are clearly arranged in four rows of three cells, while in C. hexalineatus the spermatheca cells are more randomly distributed. The spermatheca-uterus transition is indistinct in Cephalenchus, and this is especially the case in C. leptus, where the spermatheca and uterus cellular pattern are comparable. The Basiria species studied as well as Boleodorus thylactus and Neopsilenchus magnidens, (Fig. 3A-F) have a spermatheca comprised of 16 cells. The spermatheca is long and axial in Basiria while always clearly offset, but it is variable in shape in Boleodorus thylactus. In Basiria, the spermatheca is connected to the uterus by two cells. The axial spermatheca of Neopsilenchus magnidens (Fig. 3F) shows a remarkable variation in length, ranging from short and oval-shaped to long and cylindrical; the number of cells does not vary with size. In Psilenchus aestuarius (Fig. 3G), the spermatheca comprises a long axial sac of 18 to 20 cells; the spermatheca cells are elongated except for two more distal-proximally attened cells that connect spermatheca to uterus. Belonolaimidae (Fig. 4): The oviduct and uterus structure is comparable in all examined Belonolaimidae species. The oviduct consists of two rows of four cells; however, in several specimens the oviduct cells are difficult to observe since they are compressed between ovary and spermatheca. The uterus comprises three distinctive rows of large columnar cells. Each row encompasses four to ve cells in Geocenamus nothus, G. brevidens, G. microdorus, Tylenchorhynchus dubius, N. obscurus: ve to seven cells in G. cf. nurserus, G. quadrifer, T. ventralis and T. microphasmis, and seven to nine cells in T. maximus. The spermatheca within the Belonolaimidae is axial to offset, spherical to distinctly lobed in shape, and the number and arrangement of its cells is species specic. The offset spermatheca of G. quadrifer (Fig. 4A) is composed of 12 cells, variable in arrangement, with the two distal cells in continuation with the oviduct cell rows. The axial spermatheca of G. cf. nurserus and G. brevidens (Fig. 4B,C) consists of 12 to 14 cells, and cell boundaries are highly meandering in G. cf. nurserus (Fig. 4C). The spermatheca of G. nothus (Fig. 4D) shows four lobes, which are more pronounced in the presence of sperm; two cells, often with unclear cell boundaries, build up each offset lobe. More proximal, six cells constitute the axial part of the spermatheca; the two largest cells are positioned centrally, while two to four smaller cells are connected with the uterus. The spermatheca of Tylenchorhynchus dubius (Fig. 4E,K) usually comprises 12 cells of equal size and in variable position (14 cells observed in a few species); a characteristic bend occurs at the junction of the spermatheca and uterus. The spermatheca of T. microphasmis (Fig. 4G) consist of two compartments; six slightly protruding cells form a wider distal component and six to eight slightly smaller cells, of which two cells connect spermatheca with uterus, constitute a proximal component. The spermatheca of T. ventralis (Fig. 4H) comprises 12 to 14 cells, with two distal cells connecting to the oviduct and two proximal cells connecting to the uterus; the remaining cells are variable in shape and arrangement. The spermatheca of T. maximus (Fig. 4I) consists of only eight to 10 cells with indistinct cell boundaries; two relatively large cells, resembling uterus cells, connect spermatheca to uterus. The spermatheca of Nagelus obscurus (Fig. 4J) is offset and comprises 12 to 14 cells with distinctly crenate cell boundaries; two large cells form the connection to the uterus. The spermatheca outline of Amplimerlinius icarus (Fig. 4F) is bell-shaped. The cellular architecture is variable; 12 to 14 cells of inconsistent shape and size have crenate or smooth cell boundaries. Hoplolaimidae (Fig. 5): The oviduct of the examined Hoplolaiminae species persistently comprises two rows of four cells, which can be difficult to discern since the oviduct is often compacted between ovary and spermatheca. The uterus consists of three cell rows, each row being four to ve cells long. The spermatheca of Helicotylenchus comprises 12 cells; the extent to which the spermatheca is offset differs within the species studied. The spermatheca of H. cf. dihystera and H. pseudorobustus (Fig. 5 A,B) (from the latter only a limited number of extruded gonoducts were available) is only partially offset; the spermatheca cell boundaries of H. cf. dihystera are slightly crenate. H. varicaudatus and H. canadensis (Fig. 5 C,D) have a maximally offset spherical spermatheca; the proximal oviduct cells reach the distal uterus cells. Helicotylenchus pseudorobustus and H. cf. dihystera are characterized by a pair of cells, situated between spermatheca and uterus, that are larger than spermatheca cells but smaller than uterus cells. The spermatheca of the Rotylenchus species studied (Fig. 5E,F,H) likewise comprises 12 cells, except 14 cells in one dissection. The arrangement of the spatial spermatheca cell of R. uniformis is variable, while in R. goodeyi two distal spermatheca cells usually connect with the oviduct, and two proximal spermatheca cells connect with the uterus. Two rows of three smaller cells can form a sphincter-like structure between the tricolumella and uterine sac (observed in Rotylenchus goodeyi

Tylenchoidea gonoduct: Bert et al. 369

Fig. 3. The cellular architecture of oviduct, spermatheca and distal part uterus of Boleodorinae. A) Basiria gracilis. B) B. graminophila from dumpsite. C) B. graminophila from potato eld, including LM photographs. D) B. duplexa. E) Boleodorus thylactus from botanical garden. F) Neopsilenchus magnidens. G) Psilenchus aestuarius. ova.: proximal end of ovary; ovi.: oviduct; sp.: spermatheca; ut.: uterus. Scale bars = 10 m.

(Fig. 5E)). Scutellonema bradys (Fig. 5G) has an axial spermatheca comprising 12 to 14 cells with unclear cell boundaries. The spermatheca of Hoplolaimus aegypti (Fig. 5H) is weakly differentiated from the gonoduct and consists of nine to 12 cells; the exact number of cells could not be determined since the spermathecauterus transition is not clear-cut. The nature of several cells occurring between spermatheca and tricolumella

is not obvious for H. aegypti; these cells show an uteruslike pattern but are more compact (only a limited number of gonoducts were examined, n = 4). The oviduct of Rotylenchulus reniformis, which represents the Rotylenchulinae in this study, consists of two rows of four cells. The remaining cellular gonoduct architecture could not be unambiguously determined since the cell boundaries appeared indistinctly and only

370 Journal of Nematology, Volume 38, No. 3, September 2006

Fig. 4. The cellular architecture of oviduct, spermatheca and distal part uterus of Belonolaimidae. A) Geocenamus quadrifer. B) G. brevidens, including LM photographs. C) G. cf. nurserus. D) G. nothus from canal bank Lokeren, including LM photographs. E) Tylenchorhynchus dubius from canal bank Roeselare-Leie. F) Amplimerlinius icarus from apple orchard. G) T. microphasmis. H) T.ventralis. I) T. maximus. J) Nagelus obscurus. K) T. dubius from corn eld. ova.: proximal end of ovary; ovi.: oviduct; sp.: spermatheca; ut.: uterus. Scale bars = 10 m.

a limited number of gonoducts were successfully extruded. The spermatheca apparently comprises 12 to 17 variably arranged cells. The spermatheca-uterus transition is indistinct, and the uterus cells appear to be arranged in three unclear rows. Meloinema (Fig. 5I): The gonoduct structure of Meloinema is similar to that of the Belonolaimidae and Hoplolaimidae: the oviduct consists of two rows of four

cells, an oval spermatheca is comprised of 12 to 14, and uterus cells are arranged in three rows. However, each row does not consist of a limited number of cells, but the exact number of cells in this elongated uterus could not be determined. Meloidogynidae, ultrastructure of oviduct-spermatheca region of Meloidogyne incognita: Previous LM results have shown that the gonoduct of the genus

Tylenchoidea gonoduct: Bert et al. 371

Fig. 5. The cellular architecture of oviduct, spermatheca and distal part uterus of Hoplolaimidae and Meloinema. A) Helicotylenchus cf. dihystera. B) H. pseudorobustus. C) H. varicaudatus. D) H. canadensis. E) Rotylenchus goodeyi. F) Scutellonema bradys. G) Hoplolaimus aegypti. H) R. uniformis. I) Meloinema odesanens. ova.: proximal end of ovary; ovi.: oviduct; sp.: spermatheca; ut.: uterus; con.: constriction between uterus and uterine sac. Scale bars = 10 m.

Meloidogyne typically consists of an oviduct that is comprised of two rows of four cells; its spherical spermatheca is composed of characteristic lobe-like protruding cells that have often interlaced cell boundaries; and the uterus cells are arranged in three elongated cell rows (see Bert et al., 2002). As seen by TEM, two facing oviduct cells are connected to each other only for 4 to 5 m; adhering junctions strengthen the con-

nection (Fig. 6B,C). The lumen of the oviduct is extremely narrow (Fig. 6C). The oviduct cells are lled with lipid droplets, strongly stained lamellar bodies, and rough endoplasmic reticulum. Mitochondria are concentrated around the variably shaped nucleus. The oviduct cell membrane displays several invaginations. The cytoplasm of the spermatheca cells is distally densely lled with rough endoplasmic reticulum, and

372 Journal of Nematology, Volume 38, No. 3, September 2006

Fig. 6. TEM study oviduct-spermatheca region of Meloidogyne incognita. A) Overview drawing based on LM of expelled gonoduct. B) Transversal section through oviduct. C) Detail lumen oviduct. D) Oblique section through spermatheca and distal end of uterus. E) Detail proximal region spermatheca cell with indication of trans-membranous transport. F) Detail base of spermatheca cell lobe. cellb: cell boundaries of two adjacent spermatheca cells; mit: mitochondria; n: nucleus; nl: nucleolus, rer: rough endoplasmic reticulum. (Scale bars: A = 30m; B = 1m; C = 500 nm; D = 2 m; E and F = 500 nm)

vesicular bodies are in connection with the outside of the cell (Fig. 6D,E). At the base of each lobe, the connection between two adjacent cells is sinuous (Fig. 6F). Microtubules connect to the cell membranes in this region (not shown). The spermatheca has a wide lu-

men, and the spermatheca wall can be less than 1 m wide at the base of the lobes. The spermatheca nuclei and nucleoli are similar to the oviduct nuclei and nucleoli. The uterine wall is relatively wide, even in the presence of an egg (uterus not shown). The uterus cell

Tylenchoidea gonoduct: Bert et al. 373 nuclei each contain a large round nucleolus that almost completely lls the nucleus. The uterus cytoplasm is dense, rich in mitochondria and endoplasmic reticulum. Discussion Females of the Tylenchidae taxa possess an oviduct with two rows of three to seven cells; a spermatheca may or may not be offset and is comprised of 10 to 20 cells; the uterus cells are arranged in four rows. Based on a limited number of populations studied, the spermatheca cellular morphology, and more particularly the number of cells in the offset part of the spermatheca, can provide an additional tool to characterize species within the genus Tylenchus. For example, the spermatheca of T. arcuatus is not offset, while T. elegans has an offset part of four cells and in T. davainei ve to seven cells compose the offset part. Unlike the results of Brzeski (1999), who suggested the synonymization of T. davainei and T. elegans, a minimal but consistent difference in spermatheca cell composition between these species provides an additional argument to maintain the two as valid species. Conversely, the Filenchus populations apparently show relatively high intraspecic variability of the gonoduct. Filenchus species are particularly difficult to characterize, and the study of additional morphological gonoduct attributes does not solve this problem. Only F. quartus appears to be consistently different from the other described Filenchus species in having an offset spermatheca pouch. Remarkably, the actual presence of a spermatheca can not always be discerned from in toto material; when a spermatheca appears to be absent or is described as being absent, a distinct spermatheca is always manifest after dissection. This is in disagreement with the results of Chizhov and Berezina (1988a), who noted a negative correlation between tail length and development of spermatheca for their studies of Filenchus species. The cell composition of the gonoduct in Coslenchus (with exception of C. costatus) and Aglenchus (Atylenchinae Skarbilovich, 1959) is comparable with that observed in Filenchus and Tylenchus (Tylenchinae). Conversely, the oviduct and spermatheca structure in Basiria, Boleodorus and Neopsilenchus is different by the presence of a slightly helical oviduct consisting of two rows of ve cells and a spermatheca comprised of 16 cells. These gonoduct attributes appear characteristic for the Boleoderinae Khan, 1964. Although the oviduct and the spermatheca outlines resemble those of Boleodorinae, Psilenchus aestuarius differs in having a paired genital system and a uterus containing more than 35 irregularly organized cells. Considering the didelphic gonoduct arrangement and presence of phasmids on the tail, Ryss (1993), Sturhan and Rahi (1996) and Siddiqi (2000) stated that the placement of Psilenchus into a separate, though obviously paraphyletic, taxon (Psilenchidae in Dolichodoroidea, see Siddiqi, 2000) appeared more justied compared with the classication of Psilenchus in the Tylenchidae (Geraert and Raski, 1987). According to the current study, the absence of a clear quadricolumella, or tricolumella, does not allow one to assign the position of Psilenchus to either Tylenchidae or Dolichodoroidea on the basis of this character alone. The Cephalenchus species studied (Tylodorinae Paramonov, 1967) are distinguished by their elongated oviduct, which comprises ve to seven cells in each of the two rows of oviduct cells; this is higher than hitherto reported for the Tylenchoidea. Other Tylodorinae (Eutylenchus, Cephalenchus, Campbellenchus and Tylodorus) have been characterized by the spatial sequence of an elongate spermatheca followed by a transition zone of several cells, ve or six cells in each row of the crustaformeria-part of the uterus, a second transition zone of several cells and a long uterine sac (in toto observations by Geraert and Raski, 1987). Belonolaimidae and Hoplolaimidae show an overall similarity in the spatial arrangement of the gonoduct cells: two rows of four cells constitute the oviduct; the spermatheca comprises eight to 14 (mainly 12) cells; and the uterus is composed of three cell rows (tricolumella). The highest diversity is observed in the number and spatial arrangement of the spermatheca cells, especially within the genera Geocenamus and Tylenchorhynchus. The spermatheca outline in Geocenamus species (based on in toto observations) has previously been used in descriptions. For example, G. (Merlinius) nothus and G. (Merlinius) microdorus are known to have a bilobed spermatheca when the spermatheca is lled (Brzeski, 1998). However, closer examination after dissection can rene these species delimitations. The spermatheca of G. nothus comprises four lobes, while G. microdorus comprises only two lobes (independent of the presence or absence of sperm). The spermatheca of G. (Scutylenchus) quadrifer resembles the spermatheca of H. canadensis and H. varicaudatus, while the spermatheca of Geocenamus cf. nurserus is distinctive by the highly indented spermatheca cells. The spermatheca of T. (Bitylenchus) dubius (1214 equally sized cells) is clearly different from the spermatheca of T. (Bitylenchus) maximus (eight-10 cells, two large cells proximally) and more similar to the spermatheca of T. (Telotylenchus) ventralis (1214 cells, cells rather equally sized). This noticeable spermatheca diversity contradicts Geraert (1981), who reported a constancy of the spermatheca structure at the generic level for the Tylenchomorpha. However, the genus Geocenamus is considered here in a broad sense (following Fortuner and Luc, 1987), and the question arises if the gonoduct structure supports the subdivision of Geocenamus as advocated by Siddiqi (2000). Yet, the spermatheca diversity observed in our study does not correspond with known subdivisions within the genera Geocenamus and

374 Journal of Nematology, Volume 38, No. 3, September 2006 Tylenchorhynchus. However, our data do not permit corroboration of any alternative for current generic denition. Nevertheless, the spatial cellular arrangement of the spermatheca provides valuable information to characterize species within the genera Geocenamus and Tylenchorhynchus. The spermatheca of Helicotylenchus and Rotylenchus shows less variation; their 12-celled spermatheca structure appears to be stable, conrming former observations (Geraert, 1981). The gonoduct structure described in this study for the Belonolaimidae, Hoplolaiminae, Rotylenchulinae and Meloinema is analogous to that of the Pratylenchidae (with exception of Nacobbus aberrans) and the Heteroderinae. The characteristic elongation of the three uterus cell rows in Meloinema is an attribute shared with the Heteroderidae and Meloidogyne (Bert et al., 2002, 2003). Although the genus Meloinema is classied in Meloidogynidae, its spermatheca is clearly hoplolaimidlike and does not share the typical Meloidogyne characteristics, namely, spherical in shape and with lobe-like protruding cells. Further phylogenetic analyses including molecular data are necessary to clarify the position of Meloinema. Our results obtained for M. incognita agree well with the observations of the ultrastructure of the gonoduct in M. javanica (McClure and Bird, 1976). We refer to these authors for a more extended discussion about the morphological and functional aspects of the Meloidogyne gonoduct. The morphology of the oviduct was described as a remarkably stable structure within the tylenchs (Geraert, 1983). Our expanded LM results do not deviate from this, without exception, with respect to the oviduct being comprised of two rows of four cells. The very narrow lumen between the two rows of tightly packed oviduct cells shows clearly that the oviduct is a constriction between ovary and spermatheca. In other nematodes, e.g., Xiphinema theresiae and X. meridianum (Van de Velde et al., 1990a, 1990b), the complete absence of an apparent lumen is known. For X. meridianum, it is assumed that the oviduct cells are separated and pushed aside when an oocyte passes through, this because two or three nuclei are observed at the same level and the cell membranes of neighboring cells are highly intertwined (Van de Velde et al., 1990a). In Meloidogyne, oocytes pass through the oviduct in a clearly different way. Most likely, the oviduct lumen stretches considerably, and adhering junctions prevent the separation of two adjoining oviduct cells during this process. Ultrastructural analysis of the spermatheca substantiates that the Meloidogyne spermatheca is distinctive from that in any other currently known nematode genus (Bert et al, 2002), and that the spermatheca of M. incognita is an exceedingly complex structure. The outer membranes are distinctly invaginated and surround densely packed rough endoplasmic reticulum. This increased surface area could function in the transfer of metabolites, and thus the spermatheca probably has a more complex role than that of a simple receptacle. TEM of Meloidogyne was used in this study as a test case to further evaluate our LM observations of expelled gonoducts. The latter, relatively easy technique preserves the three-dimensional structure well. However, with light microscopy some aspects of interpretation may be speculative because morphological discrimination between the cells of adjacent gonoduct parts can be difficult. On a subcellular level, and with the aid of TEM, these morphological differences are much more evident. Based on our limited TEM data, ultrastructural information does conrm LM observations. Consequently, it seems that the LM-based interpretation of the gonoduct components is justied. The combination of expelled organs and TEM offers valuable possibilities. When a TEM study of a specic organ is required, it is labor saving to perform this study after expelling the organ of interest. More indirectly, TEM studies can be combined with LM studies of expelled organs to retain the spatial overview. This is especially useful if only a limited number of sections are studied by TEM. The combination of TEM on selected sections related to the knowledge of the cellular structure based on expelled organs is a relatively uncomplicated method that combines three-dimensional knowledge with ultrastructural detail. We conclude that the information associated with the cellular gonoduct structure offers promising results for diagnostic purposes, as well as for analyzing relationships. Therefore, we advocate the description of the gonoduct morphology as a valuable component in the (re) description of (new) nematode species. Further, gonoduct data, together with other morphological data and molecular data, need to be assessed to infer phylogenetic relationships, especially in Tylenchina. Literature Cited
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